Generation of Replication Competent Viruses for Therapeutic Use

ABSTRACT

The present invention relates to the generation or replication-competent viruses having therapeutic utility. The replication-competent viruses of the invention can express proteins useful in the treatment of disease.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/632,671, filed on Dec. 1, 2004, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to the generation of replication competentviruses having therapeutic applications.

BACKGROUND OF THE INVENTION

The goal of gene therapy is to deliver genetic material of therapeuticvalue to a target tissue in a safe and efficient manner. Safety is oftenrelated to how much damage is done to the normal tissues of the patientduring treatment. Efficiency can be looked at as a ratio of desiredresult, e.g. reduction of tumor load, to acceptable dosage level, wherethe parameters which contribute to making a dosage level “acceptable”can include issues of injection volume, frequency, etc. Therefore, anyimprovements that result in an increase in the selectivity andefficiency of gene therapy are clearly desirable.

Viral vectors derived from adenoviruses, have been the most studieddelivery agents for this type of therapy (Jolly, D. (1994) Cancer GeneTherapy 1:51-64). Replication-defective vectors are limited in theirusefulness due to their ability to only kill tumor cells that have beendirectly infected with virus. Viral vectors developed from oncolytic(i.e. replication-competent) viruses are more attractive choices forcancer therapeutics because they not only selectively target tumorcells, but they can, through replication within the infected tumorcells, amplify and spread the input dose of infective virus throughoutthe tumor cell mass.

The potential use of vectors derived from oncolytic viruses, such asadenovirus, in gene therapy can be further increased by “arming” theviruses with therapeutic transgenes, i.e. engineering them to containtherapeutic proteins or other molecules whose in vivo expression canimpact tumor survival (Hermiston, T. (2000) J. Clin. Inv.105:1169-1172). The combination of viral replication within tumor cellsand the activity of the therapeutic molecule expressed within the cellscan provide a synergistic assault on a tumor.

The incorporation of therapeutic transgenes into an oncolytic virus is acomplex process. The insertion event needs to occur in a site thatmaintains the replication competence of the viral agent, which iscomplicated as viruses maximize their coding capacity by generatinghighly complex transcription units controlled by multiple promoters andalternative splicing (Akusjarui and Stevenin (2003 Curr. Top. Microbiol.Immunol. 272:253-286). Consequently, the choice of insertion sites fortherapeutic genes has been limited primarily to regions known to benon-essential for viral DNA replication in vitro (Hawkins et al. (2001)Gene Ther. 8:1123-1131; Kurihara et al. (2002) J. Clin. Invest106:763-771) or by the replacement of a deleted region of the viralgenome to create the oncolytic virus (Freytag et al. (1998) Human GeneTher. 9:1323-1333; Lee et al. (2001) Cancer Gene Ther. 8:397-404). Whilethese approaches allow for therapeutic gene insertion and expression,they are dependent upon a high level of understanding of the viralbiology (i.e. sites non-essential for viral replication), a known viralgenome sequence (for use in genetic engineering or utilization ofendogenous restriction enzyme sites) and the presence of molecularbiology systems for genomic manipulations that may not be currentlyavailable for non-Ad5-based systems.

In view of the above, there is a need for a method for generatingreplication competent viruses which contain genetic elements, e.g., agene which encodes a therapeutic protein or RNA, positioned within theviral genome such that expression of the genetic element occurs. Ofparticular utility would be a method that identifies functionalinsertion sites within replication competent viruses whose genomicstructure has not yet been elucidated.

SUMMARY OF THE INVENTION

The present invention provides a novel method for identifying functionalinsertion sites within the genome of a replication competent virus andfor generating replication competent viruses which comprise a geneticelement of interest or an expression cassette inserted in one of theidentified insertion sites.

In particular, the method of the present invention comprises the stepsof

-   -   (a) mixing the genomic DNA of a replication competent target        virus with a donor DNA comprising a transposon under conditions        that allow transposition, wherein said transposon comprises at        least one genetic element of interest and inserts into said        target viral genome in a non-biased manner; and    -   (b) isolating replication competent product viruses from said        step (a) which express said genetic element of interest.

In one embodiment of the present invention, the replication competenttarget virus is an animal virus, preferably an oncolytic animal virus.In a preferred embodiment, the animal virus is an adenovirus, VSV, NDV,HSV or vaccinia virus. Particularly preferred are adenoviruses belongingto groups B and C. A particularly preferred Group C adenovirus is Ad5.

In one embodiment of the present invention, the genomic DNA of thereplication competent target virus is present within a plasmid. In apreferred embodiment, the genomic DNA of the replication competenttarget virus within the plasmid is flanked by restriction enzyme sites.Particularly preferred is an embodiment in which the restriction enzymesites flanking the genomic DNA of the replication competent target viruswithin the plasmid are identical.

In one embodiment of the invention, the donor DNA comprising thetransposon is a plasmid and further comprises an origin of replication.A preferred origin of replication is the R6K origin of replication.

In one embodiment of the present invention, the transposon comprises agenetic element of interest that is a gene that encodes a reporterprotein, where the reporter protein includes, but is not limited to,green fluorescing protein (GFP), LacZ, renilla luciferase or fireflyluciferase. Particularly preferred is a gene encoding GFP.

In another embodiment of the present invention, the genetic element ofinterest is a gene that encodes a therapeutic protein, where thetherapeutic protein can be, but is not limited to, an immunomodulatoryprotein, an antibody, a symporter, a pro-drug converting enzyme, afusogenic glycoprotein, or fragments thereof. Preferred immunomodulatoryproteins include molecules identified as cytokines or chemokines.

In another embodiment of the present invention, the genetic element ofinterest encodes a therapeutically useful RNA molecule including, butnot limited to, shRNA or antisense RNA.

In one embodiment of the present invention, the transposon furthercomprises an expression element located upstream from, and operablylinked to, the genetic element of interest, such that the expressionelement and the genetic element of interest comprise an “expressioncassette”. In a preferred embodiment, the expression element is aeukaryotic promoter. Preferred promoters include, but are not limitedto, traditional Pol II promoters (e.g. E2F or hTERT), Pol III promoters(e.g. U6 or H1) or viral promoters (e.g. CMV, SV40, HSV TK or Ad MLP). Aparticularly preferred promoter is the early SV40 promoter.

In another embodiment of the present invention, the expression elementis a eukaryotic splice acceptor sequence. Particularly preferred spliceacceptor sequences include SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.Particularly preferred is the splice acceptor sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the transposon furthercomprises restriction enzyme sites unique to the transposon, i.e. notpresent in the genomic DNA of the replication competent target virus,which are located close to the ends of the transposon. In a preferredembodiment, the restriction enzyme sites are identical. A particularlypreferred restriction enzyme site is a Pmel site.

In one embodiment of the present invention, the transposon is one thatemploys an ATP-dependent utilizing regulatory protein in thetransposition process. Particularly preferred is a Tn-7 basedtransposon.

In another embodiment of the present invention, the transposon furthercomprises a selectable/identifiable gene, such as a gene that confersdrug resistance or one that provides a mechanism for visualidentification of cells. Preferred genes that confer drug resistanceinclude genes that confer antibiotic resistance. Particularly preferredis a gene that confers chloramphenicol resistance. Among the genes thatallow for visual identification of cells are those encoding GFP, renillaluciferase, or firefly luciferase.

The present invention also provides for replication competent virusesmade by the methods described herein and for the use of these viruses asvectors for the delivery of therapeutic molecules during gene therapy.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings.

FIG. 1. Schematic of transposon-based method to identify novel insertionsites within the genome of a replication competent virus. A donorplasmid containing both an R6K origin of replication and a transposon ismixed with a plasmid (pCJ51) containing the Ad5 genome (with the E3region deleted) flanked by two Pacl restriction enzyme sites. Thetransposon contains a reporter gene, GFP, operably linked to an earlySV40 promoter, i.e. an SV40/GFP expression cassette, and also contains achloramphenicol resistance gene (CM^(R)). Following in vitrotransposition (see Example 3), E. coli DH10B cells are transformed withthe transposition mixture, and the transformed cells plated onchloramphenicol-containing plates. The donor plasmid, because of its R6Korigin of replication, cannot grow in DH10B cells. Plating onchloramphenicol-containing media selects for those cells containing Ad5plasmids into which a transposon has been inserted. Chloramphenicolresistant colonies are pooled and plasmid DNA extracted. The extractedDNA is digested with Pacl to release linearized Ad5 genomic DNAscontaining transposons inserted into various locations within the Ad5genome. The isolated linearized Ad5 genomic DNA is transfected into acell line of choice and individual green plaques, each containing areplication competent product virus, are picked for furthercharacterization (e.g. insert site location, potency, etc.)

FIG. 2. Distribution of transposon insertions within the Ad5 genomePlasmid DNA containing inserted transposons, isolated from 60chloramphenicol-resistant colonies, was digested with Bgl II and theresulting DNA fragments were analysed on 1% agarose gels. The Bgl IIrestriction fragments of the Ad5 containing plasmid, pCJ51, range insize from 8.6 kb (which includes the entire plasmid portion) to 0.27 kb.The transposon contains a Bgl II restriction site within 32 bp of theright end of the transposon. Therefore, insertion into any Bgl IIfragment will result in loss of that fragment and the appearance of twonew fragments, the sum of which will total the fragment plus 3.1 kb (thesize of the transposon). The fragment sizes will vary depending upon thesite of insertion within the fragment, and on the orientation of theinserted transposon. The percentage of insertions within each fragmentis shown on the y-axis, while the continuous line indicates thepercentage predicted if insertion is non-biased (see Example 4).

FIG. 3. Insertion sites of SV40/GFP containing transposons within the Ad5 genome The outlined area illustrates the transcription and translationmap of Ad5. The early mRNAs are designated E. Late mRNAs are designatedL. Black ovals circle the designated “E1” and “L5 to E4” regions in thetranslation map of Ad5 and are enlarged in the map below, whichindicates the site of insertion (vertical arrow) of a transposoncomprising an SV40/GFP expression cassette within the Ad5 genome in eachof a number of product viruses (see Example 4). Horizontal arrows aboveeach product virus name indicate the transcriptional direction ofSV40/GFP expression in that virus. The open dotted line represents theremaining Ad5 genome between E1B and L5.

FIG. 4. Potency of Ad5-viral isolates containing transposons. MTT assayswere performed in HT29 cells comparing the potency of the original Ad5virus (AdCJ51) and a number of transposon-containing viruses, asdescribed below. The transposon-containing viruses, Ad5/PL11 andAd5/PL29, contain the SV40/GFP expression cassette inserted within theAd5 genome, with GFP expression oriented leftwards or rightwards in theAd5 genome, respectively (see arrows in FIG. 3, Example 4). In otherviruses tested, the original expression cassette (SV40/GFP) present inthe Ad5/PL11 and Ad5/PL29 viruses was replaced by a CMV/Luc, TK/RL orCMV/LacZ expression cassette as described in Examples 5 to 7.

Panel A: the original virus, AdCJ51 (◯); and two product viruses,Ad5/PL11/SV40/GFP (□) and Ad5/PL29/SV40/GFP(Δ), in which the SV40/GFPexpression cassette had been determined to have been inserted inopposite orientations within the viral genome (see FIG. 3);

Panel B: Ad5/PL11/SV40/GFP (□); and Ad5/PL11/TK/RL (◯); Ad5/PL11/CMV/Luc(♦) and Ad5/PL11/CMV/LacZ (♦), where the expression cassettereplacements have the same orientation within the Ad 5 genome as theoriginal SV40/GFP expression cassette; and

Panel C: Ad5/PL29/SV40/GFP (Δ →); Ad5/PL29/CMV/Luc (⋄ →,  ←));Ad5/PL29/TK/RL (▪→, ▴ ←)) and Ad5/PL29/CMV/LacZ (□ →), where theexpression cassette replacements are oriented in both directions, asindicated by the arrows.

FIG. 5. Insertion Sites of Splice Acceptor/GFP transposons within theAd5 genome The outlined area illustrates the transcription andtranslation map of Ad5. The early mRNAs are designated E. Late mRNAs aredesignated L. Black ovals circle the designated “E1” and “L5 to E4”regions of the transcription/translation map of Ad5 and are enlarged inthe map below, which indicates the site of insertion (vertical arrow) ofthese transposons. Horizontal arrows above the product virus nameindicate the transcriptional direction of the GFP expression in thatviral construct.

FIG. 6: Cytotoxicity of ColoAd1/PL30/TK/TK adenovirus with and withoutganciclovir (GCV). MTS assays to measure potency were performed usingthe ColoAd1/PL30/TK/TK virus, which was derived from the ColoAd1 virus(SEQ ID NO: 22) as described in Example 9 and contains an expressioncassette comprising a thymidine kinase gene, operably linked to athymidine kinase promoter. Potency of this viral construct was examinedin both a normal cell line (HUVEC; FIG. 6A) and in a lung cancer cellline (A549; FIG. 6B). Cytotoxicity in each cell line was determined withand without the addition of 20 uM ganciclovir, 24 hrs post infection(see Example 9).

DETAILED DESCRIPTION OF THE INVENTION

All publications, including patents and patent applications, mentionedin this specification are herein incorporated by reference to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated by reference in its entirety.

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of ordinary skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, Planview, N.Y.; McPherson, M.J., Ed. (1991) Directed Mutagenesis: A Practical Approach, IRL Press,Oxford; Jones, J. (1992) Amino Acid and Peptide Synthesis, OxfordScience Publications, Oxford; Austen, B. M. and Westwood, O. M. R.(1991) Protein Targeting and Secretion, IRL Press, Oxford. Any suitablematerials and/or methods known to those of ordinary skill in the art canbe utilized in carrying out the present invention; however, preferredmaterials and/or methods are described. Materials, reagents and the liketo which reference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures described below are those wellknown and commonly employed in the art.

As used herein, the term “replication competent virus” refers to a viruswhich contains all the information within it's genome to allow it toreplicate within a cell: i.e. it either produces the protein or inducesthe necessary host proteins.

As used herein, the term “target virus” refers to a replicationcompetent virus capable of undergoing transposition, as contemplated bythe invention. While the target virus will usually be a virus which hasnot previously undergone transposition, it is possible to use as atarget virus a replication competent virus which has already undergone around of transposition.

As used herein, the term “product virus” refers to the replicationcompetent virus produced when a “target virus” has undergonetransposition by the method of the invention.

As used herein, a “functional insertion site” within a replicationcompetent virus refers to a site within that virus where, when a geneticelement, operably linked to an expression element (i.e. a eukaryoticpromoter or eukaryotic splice acceptor sequence) is inserted into thatsite, expression can occur.

As used herein, the term “genetic element of interest” refers to anucleic acid sequence that is introduced into a transposon and thatencodes a protein or RNA molecule whose expression is useful in thepresent invention.

A genetic element of interest includes genes that encode reporterproteins, i.e. proteins that are easily assayed or identified, e.g.green fluorescent protein (GFP), renilla luciferase, or fireflyluciferase. A genetic element of interest, as used herein, alsoencompasses any gene encoding a protein, protein fragment or peptide (ormodifications thereof) for which a therapeutic purpose can beenvisioned. Examples include, but are not limited to, genes encodingimmunomodulatory proteins, antibodies, symporter, fusogenicglycoproteins or pro-drug converting enzymes.

The term “genetic element of interest” also includes genes encoding RNAmolecules, such as antisense RNA or shRNA, whose in vivo expression canresult in modulation of cellular properties (e.g. cell growth,chemotherapeutic sensitization) by altering the expression levels of atargeted cellular protein (Karkare et al. Appl. Biochem. Biotechnol.(2004) 119:1-12).

As used herein, “useful” means that a protein or RNA expressed from aproduct virus would be useful in the evaluation or identification of thereplication competent product viruses of the invention (e.g. the productof a reporter gene) or useful in therapeutic applications (e.g. theproduct of a gene encoding a therapeutic protein or an RNA).

As used herein, a “therapeutic protein” refers to a protein, proteinfragment, or peptide that would be expected to provide some therapeuticbenefit to an organism when expressed in vivo.

As used herein, the term “expression element” refers to a nucleic acidsequence located upstream from, and operably linked to, a geneticelement of interest, and that facilitates expression of the geneticelement of interest within the product virus. The expression element canbe a promoter, e.g. a traditional POL II promoter; e.g. E2F or hTERT (Wuet al. Trends in Mol. Med. (2003) 9:421-429, a POL III promoter (e.g.U6) or a viral promoter (e.g. SV40, CMV, TK or MLP). Alternatively, anexpression element can be a eukaryotic splice acceptor sequence, e.g.SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

As used herein, the term “expression cassette” refers to a nucleic acidsequence that contains both a genetic element of interest and anexpression element upstream from, and operably linked to, the geneticelement, so that when the expression cassette is inserted in the properorientation within the viral genome, the protein or RNA encoded by thegenetic element is expressed. As used herein, an expression cassettecomprising an SV40 promoter and a GFP reporter gene is represented as“SV40/GFP”.

As used herein, the term “genomic DNA of the replication competenttarget virus” refers to the viral genomic DNA into which a transposon isinserted by the method of the invention.

As used herein, the term “product virus” refers to a genomic DNA of thereplication competent target virus of the invention which has had atransposon inserted into it by the method of the invention.

As used herein, the term “non-biased” refers to the insertion of atransposon within a viral genome where the choice of insertion site isnot substantially influenced by or dependent on particular sequenceswithin the viral genome and is, therefore, considered random.

As used herein, the term “optimally expresses” or “optimal expression”refers to the characterization of a product virus comprising a geneticelement of interest, when compared to the other isolated product virusescomprising that genetic element. The most straightforward comparativemeasurement of expression of a genetic element from a product viruswould be measurement of the level of expression of the gene productencoded by that genetic element, i.e. that product virus demonstratingthe highest level of expression of the protein encoded by the geneticelement of interest would be ranked as that showing “optimalexpression”. However, also contemplated within the scope of theinvention is measurement of “optimal expression” of a gene product by aproduct virus under varying conditions such as level of tissue-specificgene expression, expression in certain microenvironments (e.g. hypoxic,glucose deprived) or other measurements which would be indicative ofrelative in vitro or in vivo potency, etc.

As used herein, the term “adenovirus”, refers to any of the 50+ humanadenoviral serotypes currently known, or isolated in the future. See,for example, Strauss, “Adenovirus infections in humans,” in TheAdenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596(1984). These serotypes are classified in the subgroups A-F (see, Shenk,“Adenoviridae: The Viruses and Their Replication,” in Fields Virology,Vol. 2, Fourth Edition, Knipe, ea., Lippincott Williams & Wilkins, pp.2265-2267 (2001)

As used herein, the term “potency” refers to the lytic potential of avirus and represents its ability to replicate, lyse, and spread. For thepurposes of the instant invention, potency is a value which compares thecytolytic activity of a given product virus of the invention to that ofAd5 in the same cell line, i.e. potency=IC₅₀ of X/IC₅₀ of Ad5, where Xis the particular product virus being examined and wherein the potencyof Ad5 is given a value of 1.

As used herein, the term “oncolytic virus” refers to a virus thatpreferentially kills cancer cells as compared with normal cells.

As used herein, the term “therapeutic index” or “therapeutic window”refers to a number indicating the oncolytic potential of a given virusand is determined by dividing the potency of the virus in a cancer cellline by the potency of the same virus in a normal (i.e. non-cancerous)cell line.

As used herein, the term “modified” refers to a molecule with anucleotide or amino acid sequence differing from a naturally-occurring,e.g. a wild-type nucleotide or amino acid sequence. A modified moleculeretains the function or activity of a wild-type molecule; i.e. amodified product virus may retain its oncolytic activity or therapeuticbenefit. Modifications include mutations to nucleic acids as describedbelow.

As used herein, “mutation” with reference to a polynucleotide orpolypeptide, refers to a naturally-occurring, synthetic, recombinant, orchemical change or difference to the primary, secondary, or tertiarystructure of a polynucleotide or polypeptide, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide). Mutations include such changesas, for example, deletions, insertions, or substitutions.Polynucleotides and polypeptides having such mutations can be isolatedor generated using methods well known in the art.

As used herein, the term “therapeutically effective dose” or “effectiveamount” refers to that amount of product virus that ameliorates thesymptoms or conditions of a disease. A dose is considered atherapeutically effective dose in the treatment of cancer or itsmetastasis when tumor or metastatic growth is slowed or stopped, or thetumor or metastasis is found to shrink in size, so as to lead to anextension in life-span for the subject. For other disease states,appropriate endpoints for defining a therapeutically effective dosewould be within the knowledge of one skilled in the art.

Generation of Viruses of the Invention

The present invention provides a novel method for identifying functionalinsertion sites within the genome of a replication competent targetvirus and for generating replication competent viruses (“productviruses”) which comprise a genetic element of interest or an expressioncassette inserted in one of the identified insertion sites. The productviruses of the invention can express the inserted genetic elementfollowing infection of cells in vitro and in vivo.

The method for identifying functional insertion sites within thereplication competent viruses of the invention comprises mixing thegenomic DNA of a replication competent target virus with a donor DNAunder conditions that allow formation of replication competent productviruses comprising said transposon, where the donor DNA comprises atransposon capable of inserting into the genome of the target virus in anon-biased manner, and where the transposon comprises a genetic elementof interest or an expression cassette.

The replication competent product viruses can be evaluated to determinetheir utility, using assays that directly measure protein expression orthat measure an activity of the inserted genetic element. A comparisionof such values within a group of isolated replication competent viruses,containing insertions at different positions within the viral genome,allows selection of the particular product virus best suited for a giventherapeutic situation.

Genomic DNA of the Replication Competent Target Virus.

The genomic DNA of the replication competent target virus of the presentinvention can be that of any desired virus, preferably an animal virus(i.e. a virus that can replicate in an animal or an animal cell) and ismost preferably an oncolytic animal virus including, but not limited to,adenovirus, VSV, HSV or vaccinia virus. An important advantage of thepresent invention is that it allows one to use the genomic DNA of avirus for which specific prior knowledge regarding viral genomicorganization or sequence is not available. A preferred genomic viral DNAfor use in the present invention is that of an adenovirus, andparticularly preferred is an adenovirus from Groups B or C. Aparticularly preferred adenovirus within Group C is Ad5.

Wild-type viruses, as well as viral derivatives containing deletions ofnon-essential polynucleotides within the viral genome, e.g. the E3region in adenovirus (Berkner and Sharp (1983) Nucleic Acid Res.11:6003-6020) are contemplated to be useful in the present invention.Use of replication competent viruses which have a reduced viral genome,is preferred, as the total amount of DNA which can be packaged into avirus is limited by size constraints (for example, see Bett et al.(1993) J. Virol. 10:5911-5921). Therefore, use of a smaller startingviral genome (either naturally occurring or artificially made) allowsinsertion of transposons that contain larger genetic elements ofinterest or expression cassettes and also allows for the possibleinsertion of more than one transposon within the genomic DNA of thereplication competent target virus. Also useful are viral derivativeswhich have been identified as having an increased potency towardparticular cell types, such as ColoAd1 (SEQ ID NO: 22), which has beenshown to have increased potency toward colon tumor cells.

Genomic DNA of the replication competent target viruses useful in thepresent invention can be in a linear form or it can be present in aplasmid and be linearized when desired, i.e. to allow more efficienttransfection to occur (Berkner and Sharp (1983) Nucleic Acid Res.11:6003-6020). In a preferred embodiment, the genomic DNA of thereplication competent target virus is present in a plasmid, whichfacilitates initial selection and amplification of product viruses.Where the genomic DNA of the replication competent target virus ispresent in a plasmid, the viral DNA should be easily excisable from theplasmid. In one embodiment, restriction enzyme sites flank the viral DNAwithin the plasmid. In a preferred embodiment, the restriction enzymesites are identical. In a particularly preferred embodiment, the genomicDNA of the replication competent target virus is flanked by Pacl sites(see FIG. 1). Pacl, however, is only one choice of restriction enzymesite and determination of other useful restriction enzyme sites is wellwithin the knowledge of one skilled in the art.

Donor DNA

The donor DNA of the present invention, which comprises a transposon,can be in a linear form, such as a phage, or can be a plasmid.

A transposon useful in the present invention has the ability to insertitself into a viral genome in a non-biased manner, i.e. the choice ofinsertion site is not influenced by or dependent on particular sequenceswithin the viral genome. Transposons that use ATP-utilizing regulatoryproteins are contemplated for use in the invention, provided thatappropriate mutations in the ATP-utilizing protein have been made suchthat insertion is non-biased (see U.S. Ser. No. 10/024,809). Examples ofsuch transposons are Tn5090/Tn420 and Tn7. Particularly preferred is aTn7-based transposon (Biery et al. (2000) Nucleic Acid Res.28:1067-1077).

A transposon useful in the present invention comprises a genetic elementof interest.

In one embodiment, the genetic element of interest is a gene, oftenreferred to as a “reporter gene”, that encodes a detectable geneproduct, i.e. a product whose expression can be easily assayed oridentified. For instance, the reporter gene may encode a gene productthat, by enzymatic activity, gives rise to a detection signal based oncolor, fluorescence, or luminescence. Many reporter genes are known tothose of skill in the art and others may be identified or synthesized bymethods known to those of skill in the art. Useful reporter genesinclude, but are not limited to, green fluorescing protein (GFP), LacZ,renilla luciferase or firefly luciferase. A particularly preferredreporter gene is GFP. Assays for measuring these proteins are well knownin the art.

In another embodiment of the present invention, the genetic element ofinterest is a gene that encodes a therapeutic protein. A therapeuticprotein, as used herein, refers to a protein, protein fragment orpeptide, or a modification thereof, that would be expected to providesome therapeutic benefit to an organism when expressed in vivo.Therapeutic proteins contemplated in the present invention include, butare not limited to, proteins which are immunomodulatory proteins,antibodies, symporters, fusogenic glycoproteins or pro-drug convertingenzymes, and encompass any protein, protein fragment or polypeptidewhose expression within a cell would be expected, by one of skill in theart, to provide a therapeutic benefit (e.g. a cytotoxic effect, growthinhibitory effect etc.).

In one embodiment, the therapeutic protein of the present invention canbe a pro-drug activator, such as cytosine deaminase (see, U.S. Pat. Nos.5,631,236; 5,358,866; and 5,677,178). In another embodiment, thetherapeutic protein can be a known inducer of cell-death, e.g. apoptinor adenoviral death protein, or a fusion protein, e.g. fusogenicmembrane glycoprotein (Danen-Van Oorschot et al. (1997) Proc. Nat. Acad.Sci. 94:5843-5847; Tollefson et al. (1996) J. Virol. 70:2296-2306; Fu etal. (2003) Mol. Therapy 7: 48-754, 2003; Ahmed et al. (2003) GeneTherapy 10:1663-1671; Galanis et al. (2001) Human Gene Therapy 12(7):811-821).

Alternatively, a therapeutic protein of the invention can be a member ofa symporter family (e.g. sodium/iodide symporter, NIS) that would enablea therapeutic molecule to be more effectively targeted to the tumorcell.

Other therapeutic proteins, or fragments thereof, useful in the presentinvention, include those that encode immunomodulatory proteins, such ascytokines or chemokines. Examples include interleukin 2, U.S. Pat. Nos.4,738,927 or 5,641,665; interleukin 7, U.S. Pat. Nos. 4,965,195 or5,328,988; and interleukin 12, U.S. Pat. No. 5,457,038; tumor necrosisfactor alpha, U.S. Pat. Nos. 4,677,063 or 5,773,582; interferon gamma,U.S. Pat. Nos. 4,727,138 or 4,762,791; or GM CSF, U.S. Pat. Nos.5,393,870 or 5,391,485, Mackensen et al. (1997) Cytokine Growth FactorRev. 8:119-128). Additional immunomodulatory proteins include macrophageinflammatory proteins, including MCP-3. Monocyte chemotactic protein(MIP-3 alpha) may also be used.

In another embodiment, the genetic element of interest encodes a proteinwhose expression is known to enhance the ability of an oncolytic virusto eradicate the tumor, although not having any direct impact on thetumor itself. These genes include encoding proteins that (1) compromiseMHC class I presentation (Hewitt et al. (2003) Immunology 110: 163-169),(2) block complement activity, (3) inhibit IFNs and IFN-inducedmechanisms, (4) enhance NK cell based killing (Orange et al., (2002)Nature Immunol 3:1006-1012; Mireille et al. (2002) Immunogenetics 54:527-542; Alcami (2003) Nature Rev. Immunol. 3: 36-50; (5) down regulatethe immune response (e.g. IL-10, TGF-Beta, Khong and Restifo (2002)Nature Immunol. 3: 999-1005; 2002) and (6) act to breakdown theextracellular matrix and enhance spread of the virus within the tumor(e.g. metalloproteinases) (Bosman and Stamenkovic (2003) J. Pathol.2000: 423-428; Visse and Nagase (2003) Circulation Res. 92: 827-839).

In another embodiment of the invention, the genetic element of interestencodes a protein that can provide a safety mechanism, such thatexpression of the protein can be used to abort viral infection whennecessary, e.g. the Herpes Simples Virus (HSV) thymidine kinase, which,when expressed in the presence of ganciclovir (GCV), converts GCV into atriphosphate which is toxic to both cellular and viral replication (seeExample 9; FIGS. 6A and B).

In a different embodiment, the genetic element of interest comprisingthe transposon encodes a therapeutically useful RNA molecule, i.e. shRNA(Dorsett and Tuschl (2004) Nature Rev. Drug Disc. 3:318-329) orantisense RNA, which hybridizes to a coding mRNA nucleic acid sequence,e.g., a cancer protein sequence. Expression of these types of moleculeswithin a tumor cell can provide a therapeutic benefit by reducing thetranslation and/or stability of the targeted mRNA. In mammalian cells,short, e.g., 21 nucleotide, double stranded small interfering RNAs(shRNA) have been shown to be effective at inducing an RNAi response(Elbashir, et al. (2001) Nature 411:494-498). This mechanism may be usedto down-regulate expression levels of identified genes, e.g. fortreatment of or validation of relevance to disease. Antisensepolynucleotides can comprise naturally-occurring nucleotides, orsynthetic species formed from naturally-occurring subunits or theirclose homologs (see Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor,Inc., Natick, Mass.). RNA molecules of interest, include but are notlimited to, shRNAs or antisense RNA molecules which hybridize to thenucleotide sequences encoding cell survival proteins overexpressed incancer cells (e.g. survivin and XIAP (Ling and Li (2004) Biotechniques36:450-460; McManus et al. (2004) Oncogene 23:8105-8117) or multi-drugresistance genes (e.g. Stege et al. (2004) Cancer Gene Therapy11:699-706).

In another embodiment, the genetic element of interest is a chimericgene consisting of a gene that encodes a protein that traverses cellmembranes, for example, VP22 or TAT, fused to a gene that encodes aprotein that is toxic to tumor but not normal cells (Snyder and Dowdy(2004) Pharm. Res. 21:389-393).

The transposon of the invention can further comprise an expressionelement, located upstream from, and operably linked to, the geneticelement of interest described above. The nucleic acid sequencecomprising both the genetic element of interest and the expressionelement is referred to as an “expression cassette”. An expressioncassette comprising an SV40 promoter and a GFP reporter gene, forexample, is represented as “SV40/GFP”.

In one embodiment of the present invention, the expression element is aeukaryotic promoter, capable of driving expression of the geneticelement of interest when the transposon is inserted in the properorientation within the viral genome. Possible promoters include, but arenot limited to, e.g. a traditional POL II eukaryotic promoter (e.g. E2For hTERT (Wu et al. Trends in Mol. Med. (2003) 9:421-429)), a POL IIIpromoter (e.g. U6) or a viral promoter (e.g. SV40, CMV, TK or MLP). Aparticularly preferred promoter is the SV40 promoter.

In another embodiment, the expression element is a eukaryotic spliceacceptor sequence (Senapahty et al. (1990) Methods Enzymol.183:252-278). When the transposon, comprising the splice acceptorsequence upstream from, and operably linked to, a genetic element ofinterest, is appropriately inserted into the viral genome, transcriptionof the genetic element can occur. Preferred splice acceptor sequencesare the sequences SEQ ID NO: 1 (5′-TGCTAATCTTCCTTTCTCTCTTCAGG-3′), SEQID NO: 2 (5′-TTTCTCTCTTCAGG-3′) or SEQ ID NO: 3 (5′-CAGG-3′). Aparticularly preferred sequence is SEQ ID NO: 1.

In another embodiment of the present invention, the transposon furthercomprises restriction enzyme sites located close to the ends of thetransposon and unique to the transposon, i.e. not present in the genomicDNA of the replication competent target virus. In a preferredembodiment, the restriction enzyme sites are identical. A particularlypreferred restriction enzyme site is a Pmel site. The presence of theserestriction enzyme sites allows facile replacement of the geneticelement of interest or the expression cassette present in the originallyisolated product virus (see Examples 5 and 6) and thereby permits use ofone well-characterized product virus as a backbone for the creation of aseries of viral vectors. Each viral vector, although derived from oneproduct virus, can be armed with a different expression cassette andthus be used to target treatment of different diseases.

Also contemplated within the scope of the invention are further stepswithin the method of the invention that increase the efficiency ofidentification/isolation of product viruses produced by the method ofthe invention. For example, in one embodiment of the invention, productviruses isolated from bacteria are subjected to two rounds of infectionusing different eukaryotic cells, so as to increase the number of viralDNA colonies produced (see Example 4). Such additional steps are useful,but are not necessary, to the practice of the invention.

As mentioned above, the donor DNA comprising the transposon may belinear or may be a plasmid. Where the donor DNA is a plasmid, the donorDNA may further comprise an origin of replication, for example, the R6Korigin of replication. A properly chosen origin of replication insuresthat donor plasmids will not be able to multiply in a bacterial cellchosen for amplification of product viruses. Choice of an appropriateorigin of replication is well within the knowledge of one skilled in theart.

The transposon can further comprise a marker or selectable gene that isuseful in the efficient isolation and/or identification of productviruses. Such a gene often provides a selective growth advantage; e.g.,the gene may enhance cell viability, relieve a nutritional requirement,and/or provide resistance to a drug. Any desired marker or selectablegene can be used, including, but not limited to, genes conferringantibiotic resistance (e.g. resistance to chloramphenicol, tetracycline,ampicillin, kanamycin) or drug resistance (e.g. resistance tomethotrexate or G418). The preferred selectable gene may depend on theorganism being used to isolate or amplify the product viruses, e.g.antibiotic resistance when using bacterial cells, methotrexateresistance when using eukaryotic cells. Alternatively, a marker orreporter gene may encode a gene product that, by enzymatic activity,gives rise to a detection signal based on color, fluorescence, orluminescence. Many reporter genes are known to those of skill in the artand others may be identified or synthesized by methods known to those ofskill in the art. Useful reporter genes include, but are not limited to,green fluorescing protein (GFP), LacZ, renilla luciferase or fireflyluciferase. A particularly preferred reporter gene is GFP. Assays formeasuring these proteins are well known in the art. The choice of anappropriate selection/marker gene is well within the knowledge of oneskilled in the art.

In a preferred embodiment of the invention (see Example 3; FIG. 1), thedonor DNA is a plasmid comprising an R6K origin of replication and atransposon, where the transposon comprises a chloramphenicol resistancegene, as well as a GFP expression cassette, SV40/GFP, i.e. a GFP genewith an SV40 promoter operably linked to it.

Identification and Evaluation of Product Viruses.

Replication competent product viruses comprising a transposon insertedinto a functional site within the viral genome generated by the methodof the invention are identified and evaluated by measurement ofexpression of the genetic element of interest present within theinserted transposon.

Expression can be assayed in a variety of ways. If the genetic elementof interest is a reporter gene, the product encoded by the reporter geneis usually detected by an intrinsic activity associated with thatproduct. For example, the reporter gene can be chosen from fireflyluciferase (deWet et al. (1987) Proc. Natl. Acad. Sci. 1:4154-4158),bacterial luciferase (Baldwin et al. (1984) Biochemistry 23:3663-3667)or alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.182:231-238). Assays for these proteins are well known in the art andmany kits to perform these assays are commercially available (e.g.Dual-Luciferase Reporter Assay Kit, Promega, Madison, Wis.). In onepreferred embodiment, use of a reporter gene encoding GFP permitsidentification of product viruses as green-fluorescing plaques.

Other methods for measurement of gene expression that can be usedinclude, but are not limited to, Southern blotting, Northern blotting toquantitate mRNA transcription (Thomas et al. (1980) Proc. Natl. Acad.Sci. USA 77:5201-5205), DNA analysis by dot blotting, or in situhybridization, using an appropriately labeled probe, based on thesequences of the inserted genetic element. Alternatively, geneexpression may be measured by immunological methods, such asimmunohistochemical staining of cells or tissue sections, to directlyquantitate the expression of a gene product using appropriately specificantibodies. Antibodies useful for immunohistochemical staining and/orassay of cell supernatants may be either monoclonal or polyclonal.

When the genetic element of interest encodes a therapeutic protein,assays that directly measure protein expression or assays which measurean activity of the expressed protein can be used to identify productviruses of potential interest. In general, assay methods that can beused to determine levels of an expressed protein in a sample derivedfrom a host are well-known to those of skill in the art and include suchassay methods as radioimmunoassays (RIA), competitive-binding assays,western Blot analysis and enzyme-linked immunoabsorbant assays (ELISA),fluorescent activated cell sorting (FACS), and surface plasmonresonance. Among these, ELISAs frequently are preferred. An ELISA assayrequires having an antibody specific to the gene product of interest,preferably a monoclonal antibody. In addition, a reporter antibodygenerally is prepared which binds to the monoclonal antibody or isdirectly conjugated to the antibody. The reporter antibody is attachedto a detectable reagent such as a radioactive, fluorescent or enzymaticreagent, e.g. horseradish peroxidase enzyme.

To carry out an ELISA, a sample is removed from a host and incubated ona solid support, e.g. a polystyrene dish that binds the polypeptides inthe sample. Any free polypeptide binding sites on the dish are thencovered by incubating with a non-specific protein such as bovine serumalbumin. Next, the monoclonal antibody is incubated in the dish duringwhich time the monoclonal antibodies attach to any product polypeptidesattached to a solid support. Unbound monoclonal antibody is separatedfrom bound antibody by washing with buffer. The reporter antibody linkedto horseradish peroxidase is placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the geneproduct of interest. Unattached reporter antibody is then washed out.Reagents for peroxidase activity, including a calorimetric substrate arethen added to the dish. Immobilized peroxidase, linked to the productprotein through the primary and secondary antibodies, produces a coloredreaction product. The amount of color developed in a given time periodindicates the amount of product polypeptide present in the sample.Quantitative results typically are obtained by reference to a standardcurve.

A competition assay may be employed wherein antibodies specific to theproduct protein are attached to a solid support and labeled productprotein and a sample derived from the host are passed over the solidsupport and the amount of label detected attached to the solid supportcorrelated to a quantity of product protein in the sample.

These and other assays are described, among other places, in Hampton etal. (Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn., 1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

Expression of shRNA or antisense RNA expression can be measured throughmeasurement of the expression of, or activity of, the target message;i.e. a reduction in either of these measurements would indicateproduction of the desired RNA molecule.

Replacement of Genetic Element of Interest Within a Product Virus

One advantage of the present invention is the potential to use anisolated and characterized product virus of the invention as a genetherapy vector for more than one therapeutic indication. This isaccomplished by incorporating the ability to easily exchange the geneticelement of interest or expression cassette present within the transposonof the originally isolated product virus with a different geneticelement of interest or expression cassette. Transposons useful in thepresent invention, therefore, further comprise unique enzyme restrictionsites; i.e. sites not present in the starting viral genome, locatedclose to the ends of the transposon. Presence of these restrictionenzyme sites allows facile replacement of the original genetic elementof interest or expression cassette present in an isolated product viruswith a different genetic element of interest or expression cassette. Ina preferred embodiment, the restriction enzymes sites are blunt-endedrestriction enzyme sites, allowing insertion of a replacement geneticelement of interest or expression cassette in both possible orientationswithin the product viral genome. It has been shown that expressionlevels from therapeutic genes placed within the replicating virus canvary depending on the viral sequences flanking the gene and on theorientation of the gene within the viral genome (Schneider et al. (1989)J. Gen. Virol. 70: (Pt 2) 417-427; Mittal et al. (1995) Virology210:226-230) so it is useful to be able to generate product virusescontaining genetic elements (or expression cassettes) inserted in bothdirections for evaluation.

In a particularly preferred embodiment, the restriction enzyme sites areidentical. A particularly preferred restriction enzyme site is the Pmelrestriction enzyme site; however, choice of enzyme restriction sites iswell within the knowledge of one skilled in the art (see Examples 5 and6).

The replacement genetic element of interest, whether alone or within anexpression cassette can be any genetic element; however, there is alimitation on the size of the insert which can be used and still allowproper viral packaging (see, for example, Bett et al (1993) J. Virol.10:5911-5921).

Table 1 contains the results of an experiment showing replacement of anSV40/GFP expression cassette present within two isolated productviruses, identified as PL29 and PL11 (see Example 5). The SV40/GFPexpression cassette is replaced with other expression cassettes, i.e.TK/RL, CMV/LUC or CMV/LacZ (see Examples 6), allowing isolation of newreplication competent product viruses containing the expressioncassettes oriented in both directions within the PL29 and PL11 genomes.FIG. 4 demonstrates the difference in potency among the variousreplication competent product viruses generated by these substitutions.

Therapeutic Activity Measurements

Replication competent product viruses of the invention are evaluated fortheir therapeutic utility using in vitro or in vivo systems designed tomodel a particular disease or condition. Elegant models now exist forsome of the major cancer indications, e.g. prostate cancer (Russell andVoeks (2003) Methods Mol. Med. 81:89-112; Powell et al. (2003) CurrentDrug Targets 4:263-279), bone metastasis (Rosol et al. (2004) CancerTreat. Rep. 118:47-81), angiogenesis (Mg et al. (2000) J. Neurooncol.50:89-98; Hanahan et al. (1996) Eur. J. Cancer 32A:2386-2393) and thesecan be complemented, in some cases, by spontaneous tumor models in dogsand cats (Hansen and Khanna (2004) Eur. J. Cancer 40: 858-880).

Utility in the treatment of malignancy can be examined by measurement ofthe lytic potential of a product virus in tumor cells derived fromtissues of interest as therapeutic targets. Tumor cell lines useful fortesting such viruses may include, but are not limited to, colon celllines, including but not limited to, DLD-1, HCT116, HT29, LS1034 andSW48 cell lines; prostate cell lines, including but not limited to,DU145 and PC-3 cell lines; pancreatic cell lines, including but notlimited to, the Panc-1 cell line; breast tumor cell lines, including butnot limited to, the MDA231 cell line and ovarian cell lines, includingbut not limited to, the OVCAR-3 cell line. Hemopoietic cell linesinclude, but are not limited to, the Raji and Daudi B-lymphoid cells,K562 erythroblastoid cells, U937 myeloid cells, and HSB2 T-lymphoidcells. Any other tumor cell lines that are available can be used inevaluating and identifying replication competent viruses of theinvention for the treatment of neoplasia.

The cytolytic activity of the replication competent viruses of theinvention can be determined in representative tumor cell lines and thedata converted to a measurement of potency, with an adenovirus belongingto subgroup C, preferably Ad5, being used as a standard (i.e. given apotency of 1). A preferred method for determining cytolytic activity isan MTT assay (see Example 7, FIG. 4).

The therapeutic index of a replication competent virus of the inventionin a particular tumor cell line can be calculated by comparison of thepotency of the given virus in a tumor cell line with the potency of thatsame virus in a non-cancerous cell line. Preferred non-cancerous celllines are SAEC cells, which are epithelial in origin, and HuVec cells,which are endothelial in origin. These two cell types represent normalcells from which organs and vasculature, respectively, are derived, andare representative of likely sites of toxicity during viral therapy,depending on the mode of delivery of the virus. However, practice of theinvention is not limited to the use of these cells, and othernon-cancerous cell lines (e.g. B cells, T cells, macrophages, monocytes,fibroblasts) may also be used.

The replication competent viruses of the invention can be furtherevaluated for their ability to target neoplastic cell growth (i.e.cancer) by their capacity to reduce tumorigenesis or neoplastic cellburden in nude mice harboring a transplant of neoplastic cells, ascompared to untreated mice harboring an equivalent neoplastic cellburden.

Evaluation of the replication competent viruses of the invention canalso be performed using primary human tumor explants (Lam et al. (2003)Cancer Gene Therapy; Grill et al. (2003) Mol. Therapy 6:609-614), whichprovide test conditions present in tumors that cannot normally beproduced using the tumor xenograft studies.

Therapeutic Utility

The replication competent viruses generated by the method of theinvention are useful in gene therapy.

Pharmaceutical Compositions and Administration

The present invention also relates to pharmaceutical compositions thatcomprise the replication competent viruses of the invention, formulatedfor therapeutic administration to a patient. For therapeutic use, asterile composition containing a pharmacologically effective dosage ofvirus is administered to a human patient or veterinary non-human patientfor treatment, for example, of a neoplastic condition. Generally, thecomposition will comprise about 10¹¹ or more adenovirus particles in anaqueous suspension. A pharmaceutically acceptable carrier or excipientis often employed in such sterile compositions. A variety of aqueoussolutions can be used, e.g. water, buffered water, 0.4% saline,0.3%-glycine and the like. These solutions are sterile and generallyfree of particulate matter other than the desired adenoviral vector. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g. sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, etc. Excipients that enhance infection ofcells by adenovirus may be included. (see U.S. Pat. No. 6,392,069)

Viruses of the invention may also be delivered to neoplastic cells byliposome or immunoliposome delivery; such delivery may be selectivelytargeted to neoplastic cells on the basis of a cell surface propertypresent on the neoplastic cell population (e.g., the presence of a cellsurface protein which binds an immunoglobulin in an immunoliposome).Typically, an aqueous suspension containing the virions is encapsulatedin liposomes or immunoliposomes. For example, a suspension of adenoviralvirions can be encapsulated in micelles to form immunoliposomes byconventional methods (U.S. Pat. No. 5,043,164, U.S. Pat. No. 4,957,735,U.S. Pat. No. 4,925,661; Connor and Huang, (1985) J. Cell Biol. 101:581; Lasic D. D. (1992) Nature 355: 279; Novel Drug Delivery (eds.Prescott and Nimmo, Wiley, New York-, 1989); Reddy et al. (1992) J.Immunol. 148:1585). Immunoliposomes comprising an antibody that bindsspecifically to a cancer cell antigen (e.g., CALLA, CEA) present on thecancer cells of the individual may be used to target virions to thosecells (Fisher (2001) Gene Therapy 8:341-348).

Viral Therapy

The viruses of the invention, or pharmaceutical compositions thereof,can be administered for therapeutic treatment of neoplastic disease orcancer. In therapeutic applications, compositions are administered to apatient already affected by the particular neoplastic disease, in anamount sufficient to cure or at least partially arrest the condition andits complications. An amount adequate to accomplish this is defined as a“therapeutically effective dose” or “efficacious dose”. Amountseffective for this use will depend upon the severity of the condition,the general state of the patient, and the route of administration.

For example, but not by way of limitation, a human patient or non-humanmammal having a solid or haemotologic neoplastic disease, (e.g.pancreatic, colon, ovarian, lung, or breast carcinoma, leukemia ormultiple myeloma) may be treated by administering a therapeuticallyeffective dosage of an appropriate adenovirus of the invention, i.e. onewhich has been shown to have an improved therapeutic index for thattissue type. A preferred chimeric adenovirus for the treatment of coloncancer would be the adenovirus ColoAd1 (SEQ ID NO: 22). Suspensions ofinfectious adenovirus particles may be delivered to neoplastic tissue byvarious routes, including intravenous, intraperitoneal, intramuscular,subdermal, and topical. An adenovirus suspension containing about 10³ to10¹² or more virion particles per ml may be administered by infusion(e.g., into the peritoneal cavity for treating ovarian cancer, into theportal vein for treating hepatocarcinoma or liver metastases from othernon-hepatic primary tumors) or other suitable route, including directinjection into a tumor mass (e.g., a breast tumor), enema (e.g., coloncancer), or catheter (e.g., bladder cancer). Other routes ofadministration may be suitable for carcinomas of other origins, i.e.inhalation as a mist (e.g., for pulmonary delivery to treat bronchogeniccarcinoma, small-cell lung carcinoma, non-small cell lung carcinoma,lung adenocarcinoma, or laryngeal cancer) or direct application to atumor site (e.g., bronchogenic carcinoma, nasopharyngeal carcinoma,laryngeal carcinoma, cervical carcinoma).

Viral therapy using the replication competent viruses of the instantinvention may be combined with other antineoplastic protocols, such asconventional chemotherapy or x-ray therapy to treat particular cancers.Treatment can be concurrent or sequential. A preferred chemotherapeuticagent is cisplatin, and the preferred dose may be chosen by thepractitioner based on the nature of the cancer to be treated, and otherfactors routinely considered in administering cisplatin. Preferably,cisplatin will be administered intravenously at a dose of 50-120 mg/m²over 3-6 hours. More preferably it is administered intravenously at adose of 80 mg/m²over 4 hours. A second preferred chemotherapeutic agentis 5-fluorouracil, which is often administered in combination withcisplatin. The preferred dose of 5-fluorouracil is 800-1200 mg/m² perday for 5 consecutive days.

Viral therapy using the replication competent viruses of the instantinvention as vectors may also be combined with other genes known to beuseful in viral based therapy. See U.S. Pat. No. 5,648,478.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

The present invention is further described by the following examples,which are illustrative of specific embodiments of the invention, andvarious uses thereof. These exemplifications, which illustrating certainspecific aspects of the invention, do not portray the limitations orcircumscribe the scope of the disclosed invention.

Unless otherwise indicated, the practice of the present inventionemploys conventional techniques of cell culture, molecular biology,microbiology, recombinant DNA manipulation, immunology science, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, e.g. Cell Biology: a Laboratory Handbook: J. Celis(Ed). Academic Press. N.Y. (1996); Graham, F. L. and Prevec, L.Adenovirus-based expression vectors and recombinant vaccines. In:Vaccines: New Approaches to Immunological Problems. R. W. Ellis (ed)Butterworth. Pp 363-390; Graham and Prevec Manipulation of adenovirusvectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer andExpression Techniques. E. J. Murray and J. M. Walker (eds) Humana PressInc., Clifton, N.J. pp 109-128, 1991; Sambrook et al. (1989), MolecularCloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress; Sambrook et al. (1989), and Ausubel et al. (1995), ShortProtocols in Molecular Biology, John Wiley and Sons.

EXAMPLES

Standard techniques are used for recombinant nucleic acid methods,polynucleotide synthesis, and microbial culture and transformation(e.g., electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd. edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) which are provided throughout thisdocument. The nomenclature used herein and the laboratory procedures inanalytical chemistry, organic synthetic chemistry, and pharmaceuticalformulation and delivery, and treatment of patients. Methods for theconstruction of adenoviral mutants are generally known in the art. See,Mittal, S. K., Virus Res., 1993, vol: 28, pages 67-90; and Hermiston, T.et al., Methods in Molecular Medicine: Adenovirus Methods and Protocols,W. S. M. Wold, ed, Humana Press, 1999.

The following examples are offered by way of illustration and are notintended to limit the invention in any way.

Example 1 Construction of Donor Plasmids

A. Donor plasmid containing a transposon with an SV40/GFP expressioncassette. The oligonucleotides GER82(5′-AAATGTGGCCGGCCACTGATTCCACGTAGTGGTCAGGTA-3′; SEQ ID NO: 4) and GER83(5′-CTAGTACCTGACCACTACGTGGAATCAGTGGCCGGCCACATTT-3′; SEQ ID NO: 5) wereannealed and cloned into the Swal/Spel digested pGPS4 plasmid (NewEngland Biolabs, Beverly, Mass., USA), adding Fsel and Dralllrestriction sites to pGPS4 in the order Swal-Fsel-Dralll-Spel, producingthe plasmid pGER57. Annealing of oligonucleotides GER72(5′-GGAATTGGCCGGCCAT ATCCGC-3′; SEQ ID NO: 6) and GER73(5′-GGATATGGCCGGCCAATTCCGC-3′; SEQ ID NO: 7) and ligation to a Saclldigested plasmid pQBI25-fPA (Q-Biogene, Carlsbad, Calif., USA) createdplasmid pGER54, which contains a unique Fsel site immediately 5′ of thesgGFP coding sequence. The SV40 promoter/enhancer was PCR amplified fromphRL-SV40 (Promega, Madison, Wis., USA) with oligonucleotides GER94(5′-CATGGATGGCCGGCCGCTGTGGAATGTGTGTC A G-3′; SEQ ID NO: 8) and GER95(5′-TCAGTAGCTAGCCATGGTGGCTAAGAGCTGTAATTGAACTGG-3′; SEQ ID NO: 9) andrestriction enzyme digested to create a fragment with the followingfeatures: Fsel-SV40 promoter/enhancer-small IVS-Nhel. This fragment wascloned into the Fsel/Nhel digested pGER54 plasmid to create plasmidpGER59, which contains a transcription unit bounded by Fsel and Dralllin which the sgGFP gene is driven by the SV40 promoter/enhancer. Thistranscription unit was then cloned into the Fsel/Dralll digested pGER57to result in a plasmid, pGER98, which contains a chloramphenicol geneand SV40-sgGFP transcription unit within the Tn7 transposon.

Plasmids pGPS4, pGER57 and pGER98 have the R6K origin of replication andare unable to grow in ordinary lab strains of E. coli such as DH5a whichare pir− (Kolter et al. (1978) Cell 15:1199-1208 (1978); Metcalf et al.(1994) Gene 138:1-7). For transformation and growth of these plasmids, adesired amount of DNA or ligation mixture was added to 20 ulelectrocompetent E. coli TransforMax EC100D™ pir+ cells (Epicentre,Madison, Wis., USA) in a chilled microcentrifuge tube. The cell/DNAmixture was then transferred to a chilled cuvette and electroporated atthe settings recommended by the manufacturer. Immediately afterelectroporation, the cell/DNA mixture was transferred to 1 ml of SOCmedium (Invitrogen, Carlsbad, Calif., USA) and incubated at 37° C. for 1hour with shaking. The desired number of cells was plated on CM^(R) LBagar plates for growth of colonies.

B. Donor plasmids containing transposons with splice acceptor/GFPexpression cassettes. Plasmid pGER54 containing sgGFP (see above, thisExample) was digested with Fsel and Nhel, and ligated to two sets ofoligos, GER88 (5′-CCTTTCTCTCTTCAGGCCGCCATGG-3′; SEQ ID NO: 10) and GER99(5′-GTTCTGGATCCGTGAGTCAACAGGAAAGTTCC-3′; SEQ ID NO: 11), which whenannealed give a Fsel-SA-Nhel fragment, and GER100(5′-CCTGCTAATCTTCCTTTCTCTCTTCAGGCCGCCATGG-3′; SEQ ID NO: 12) and GER101(5′-CTAGCCATGGCGGCCTGAAGAGAGAAAGGAAGATTAGCAGGCCGG-3′; SEQ ID NO: 13),which when annealed give an Fsel-BPS-Nhel fragment, resulting inplasmids pGER99 and pGER100, respectively. These latter two plasmidswere digested with Fsel and Dralll and ligated to Fsel/Dralll digestedpGER57 (see above, this Example) to result in plasmids pGER111,containing a transposon with the splice acceptor sequence, SEQ ID NO: 1and pGER112, containing a transposon with the branch-point spliceacceptor sequence, SEQ ID NO: 2.

Example 2 Generation of a Starting Plasmid (pCJ51) Comprising an Ad5Genome

The left end of Ad5 was introduced into pAdEasy (Stratagene, La Jolla,Calif., USA) by homologous recombination. The Scal-BstZ17I fragment ofpTG3602 (Chartier et al, 1996) was co-transfected along withClal-linearized pAdEasy into BJ5183 bacteria (Stratagene, La Jolla,Calif., USA). The resulting recombinant plasmid was named pCJ38. TheSall fragment of pAdEasy corresponding to Ad5 nucleotides 9841-16746 wassub-cloned into the Sall site of pBluescript-KS+ (Stratagene, La Jolla,Calif. 92037, USA). The Pmel site of the resulting pCJ36 was thenmutated using the CJ23-23r pair of oligonucleotides with annealed doublestranded sequence as follows: 5′-CCGGCGGCAGAAGATCCCCTCGTTGCACA[GC*TTAAAC]AGCGAGGAGGAGCGCATTTTGCGCTA-3 (SEQ ID NO: 14). ThePmel site is bracketed and the asterisk designates the T to C pointmutation that destroys the Pmel site without disturbing the IIIa ORF.Annealed oCJ23-23r was co-transfected along with Pmel-linearized pCJ36into BJ5183 and pCJ39 was obtained by homologous recombination. Finally,the Pmel-Sall fragment of pCJ39 was re-introduced into pCJ38 byhomologous recombination into the BJ5183 bacteria after linearization ofthe latter plasmid with Pmel. The plasmid containing the Pacl-flanked,E3-deleted, and Pmel⁻ Ad5 genome was named pCJ51.

Example 3 Transposition of the sgGFP Transposon From pGER98 to pCJ51

The in vitro transposition components of the GPS™-LS Linker ScanningSystem from New England Biolabs (Beverly, Mass., USA) were utilized asdescribed in the kit, as follows. The transposon donor plasmid (pGER98,0.04 ug) was mixed with TnsABC (1 ul) and the target Ad plasmid (pCJ51,0.16 ug) and incubated for 10 minutes at 37° C. Start solution wasadded, incubated for 1 hour at 37° C., and heated at 75° C. for 10minutes, for a final volume of 20 ul. One ul of this reaction mixtureresulted in approximately 5000 chloramphenicol resistant coloniesfollowing electroporation to DH10B electrocompetent cells (Invitrogen,Carlsbad, Calif., USA).

Transposition of the splice acceptor transposons were performed in thesame way.

Example 4 Identification of Transposon Insertion Sites Within the Ad5Genome

Chloramphenicol resistant colonies (see above) were pooled (about 10,000colonies per pool) and plasmid DNA was extracted using Qiagen (Valencia,Calif., USA) endo-free plasmid extraction kits. Five ug of Pacl-digestedplasmid DNA was transfected into one 10 cm dish of HEK 293 cells by thecalcium phosphate method (Invitrogen, Carlsbad, Calif., USA). Afterovernight incubation, 15 ml of 2% agar (2% agar in 2% FBS DMEM pluspenicillin/Streptomycin) was then added to the cells. Four or five dayslater, another 10 ml of 2% agar was added. After 10 days to 3 weeksincubation, green plaques were isolated, and serial dilutions (10⁻³ to10⁻⁹) of these plaques were used to infect A549 cells plated in 6-wellplates. After 30 minutes, 2 ml of 2% agar were added to the cells. Threeto four days later another layer of agar was added, and green plaquesfrom the highest dilutions were picked and amplified (6 from 60plaques). Viral DNA was extracted from the cells using the HIRT method(Hirt B. (1967) J. Mol. Biol. 26:265-369) as follows. A549 cells wereplated in 10 cm dishes and infected with 100 ul to 200 ul of viral CPEstock after overnight culture. After 16 to 48 hours the cells were lysedwith 1 ml of HIRT extraction buffer (0.6% SDS, 0.5 nM EDTA and 1 nM TrispH 7.5) and rocked slowly in a shaker for 15 minutes, after which 250 ulof 5M NaCl was added followed by storage overnight at 4° C. The celllysate was then microcentrifuged for 30 minutes at 4° C. followed byproteinase K digestion (final concentration: 500 ug/ml) for 2-3 hours at37° C. After 1×phenol-chloroform and 1×chloroform extraction of the celllysate, viral DNA was precipitated by 2 volumes of EtOH and 1/10 volumeof 3M NaOAc pH 5.2. The DNA precipitate was then washed with 70% alcoholand resuspended in 100 ul of TE buffer.

The viral DNA was initially digested with Bgl II to identify the Bgl IIfragment containing the inserted transposon (see FIG. 2). In order todefine the exact insertion site, PCR primers capable of amplifyingapproximately 3 kb of the Ad5 genome in overlapping segments withinand/or flanking the identified Bgl II fragments were used to amplify theinserted transposons flanked by Ad5 DNA. The PCR fragments weresequenced using primers originating within the transposon and orientedtoward the Ad5 DNA at each end of the transposon. For all Ad-transposongenomes examined in this manner, the two sequences were found toindicate the identical position and orientation of the transposoninsertion for any given Ad isolate. Results are shown in FIG. 3.

Identification of the insertion sites for transposons comprising thesplice acceptor sequences SEQ ID NO: 1 (SA) or SEQ ID NO: 2(BPS) weredone in the same way (see FIG. 5).

Example 5 Replacement of Genetic Elements of Interest Within Ad5/PL11and Ad5/PL29

Two plasmids, Ad5/PL11 and Ad5/PL29, which contain replication competentAd5 viruses containing transposons comprising the SV40/GFP expressioncassette (see Example 4) were used to examine the facility with whichthe original SV40/GFP expression cassette could be replaced withdifferent expression cassettes.

A. Expression cassette construction. Three reporter gene expressioncassettes, each of which contain a reporter gene under the control of apromoter, were constructed as follows. The CMV/LacZ expression cassette(LacZ gene driven by the CMV promoter) was PCR amplified with primers(5′-AG CTGTTTAAACCGATGTACGGGCCAG-3′ (SEQ ID NO: 15) and5′-TGACGTTTAAACTAGAAGGCACAGTCGAGGC-3′ (SEQ ID NO: 16)) usingpcDNA3.1/LacZ/hygro (Invitrogen, Carlsbad, Calif., USA) as template. TheTK/RL (Renilla luciferase gene driven by TK promoter) expressioncassette was excised from the phRL/TK vector (Promega, Madison, Wis.,USA) with Bgl II and BamHI restriction endonucleases and thenblunt-ended with Klenow fragment. To construct the CMV/Luc expressioncassette (firefly luciferase gene driven by the CMV promoter), twooligonucleotides (5′-CTAGCGAATTCGGTACCACGCGTCTC GAGGCCAC-3′ (SEQ ID NO:17) and 5′-CATGGTGGCCTCGAGACGCGTGGTACCGAATTCG-3′ (SEQ ID NO: 18)) wereannealed and inserted into the pGL3Basic plasmid (Promega, Madison,Wis., USA) digested with Nhe I/Nco I, resulting in plasmid pGL3BasicIthat contains a convenient multiple cloning site (Nhe I, EcoR I, Kpn I,Mlu I, Xho I and Nco I) at the 5′ end of the firefly luciferase gene.The firefly luciferase gene was excised with Nhe I and Xba I frompGL3BasicI and cloned into Nhe I/Xba I digested phRL-CMV (Promega) toresult in the pCMV/Luc plasmid. The CMV/Luc cassette was then excisedfrom this plasmid with Bgl II and BamH I and blunt-ended for cloninginto the Pme I sites of Ad5/PL11 and Ad5/PL29.B. Expression cassette replacement. The blunt-ended expression cassettes(CM/LacZ, TK/RL or CMV/Luc) were cloned into the transposon insertionsites within the Ad5/PL11 and Ad5/PL29 viral DNA, using the Pmelrestriction sites present near both ends of the inserted transposon.HIRT extracted or CsCl purifed Ad5/pPL11 or Ad5/PL29 DNA was digestedwith Pme I to remove all but 15 bp of the 3.1 kb transposon. The threereporter expression cassettes were each blunt-end ligated toPmel-digested viral DNA using T4 DNA ligase (rapid ligation kit fromEpicentre, Madison, Wis., USA). The mass ratio of the expressioncassette fragment to viral DNA was 1:10. The ligation mixture wastransfected into HEK 293 cells using the calcium phosphate method. After10 days to two weeks, non-green viral plaques were picked and used toinfect A549 cells to test for reporter gene expression. LacZ expressionwas detected by the In Situ β-Gal Staining Kit (Stratagene, La Jolla,Calif., USA). Expression of firefly or renilla luciferase was measuredby the Dual-Luciferase reporter assay kit (Promega, Madison, Wis., USA).Reporter-gene expression positive plaques were then subjected to asecond round of plaque assays in A549 cells. Plaques were picked fromthe highest dilution wells and reporter gene expression was verified.

Example 6 Identification of Insertion Orientations of ReporterExpression Cassettes

Since Pmel is a blunt-end cutter, the blunt-ended reporter expressioncassettes can be inserted in both orientations. The orientation wasidentified by conducting two separate PCR reactions with theAd/expression cassette viral DNA as the template. A primer common toboth reactions was a reverse primer from the given expression cassette(not shown). The second primer for each reaction was one of the twoprimers from the set of Ad primers that originally identified theinsertion site (see above under “Identification of transposon insertionsites on Ad5 genome”). Only one of the PCR fragments can result in a PCRfragment, which therefore defines the direction of orientation ofinsertion of the expression cassette in the Ad genome. The results areshown in Table 1.

TABLE 1 Virus Total # Positive Orientation name clones clones → ← → 12 95 4 PL29/TK/RL 22 15 13  2 CMV/Luc 27 5  5* 0 CMVLacZ ← 12 4 0 4PL11/TK/RL 2 2 0 2 CMV/Luc 13 2 0 2 CMV/LacZ

Example 7 Viral Propagation and Potency Measurement

Human embryonic kidney cell line HEK 293 cells and human epithelial lungcarcinoma A549 cells were obtained from ATCC. Both cell lines weremaintained in DMEM with 10% FBS and 0.1 nM nonessential amino acid(NEAA). For the viral infection, DMEM with 2% FBS, 0.1 nM NEAA and 1 %penicillin/streptomycin was used. Viral propagation was performed asdescribed and viruses were quantitated using a TMAE column (Shabram etal. (1997) Human Gene Therapy 8:453) or using the Adeno-X™ Rapid TiterKit (Clontech, Palo Alto, Calif., USA. The viruses ability tosuccessfully replicate, lyse and spread was measured by using amodification of the MTT assay (Shen et al. 2003 J. Virology77:2640-2650). The MTS assay (Promega, CellTiter 96® AqueousNon-Radioactive Cell Proliferation Assay) was used in place of the MTTassay since conversion of MTS by cells into aqueous, soluble formazanreduces time and eliminates the use of a volatile organic solventassociated with the MTT assay.

To perform the assay, cells were seeded at a defined density for eachtumor cell line that generated a confluent monolayer within 24 hr. Thesedensely seeded cells were allowed to grow for 2 additional days prior toexposure to the test virus(es). Infections of both tumor and primarynormal cells were carried out in quadruplicate with serial three folddilutions of the viruses starting at a particle per cell ratio of 100and ending at a particle per cell ratio of 0.005. Infected cells wereincubated at 37° C. and the MTS assay was performed at the time pointsindicated for the individual primary cells or tumor cell lines.Mock-infected cells served as negative controls and established the 100%survival point for the given assay.

Example 8 RACE Analysis of the Splice Acceptor Clones

To determine the identity of the internal viral promoters, total RNAextracted from cells infected with selected viral clones containinginsertions of splice acceptor transposons were used as templates for PCRamplification using primers appropriate for RACE analysis. The GFP genespecific primer is 5′-GGCCATGGAACAGGCAGTTTGCCAGTAGTGC-3′ (SEQ ID NO:19). The 10 Universal Primer A Mix (UPM) used was from the BD SMART™cDNA Amplification Kit from BD Biosciences (Palo Alto, Calif.; Catalog #634914), which included primers 5′-CTAATACGACTCACTATAGGGCAAGCAGTG-3′(SEQ ID NO: 20) and 5′-TAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 21).Amplified DNA fragments were isolated from agarose gels, cloned into theappropriate vector from the kit and submitted for DNA sequencing. Theresulting sequence of the RACE fragment was compared to the knownsequence of Ad5 in order to determine those regions of the adenovirusand transposon DNA on the RACE fragments, enabling identification of thepromoter and splicing used to create mRNA containing the GFP gene.

Example 9 Generation of ColoAd1/PL30/TK/TK and its In Vitro Cytotoxicityin the Presence and Absence of Ganciclovir (GCV)

ColoAd1 (SEQ ID NO: 22) was introduced into plasmid pL30/TK/TK by themethods given in Example 2. This plasmid was then used to producereplication competent product viruses comprising a transposon containingthe expression cassette SV40/GFP. A replication competent product virus,ColoAd1/PL30, with a potency equivalent to parental ColoAd1, wasidentified and the original expression cassette replaced with a TK/TKexpression cassette, i.e. a thymidine kinase gene driven by a thymidinekinase promoter, in a manner similar to that describe in Example 5.

Human umbilical vein endothelial cells (HuVEC) and human epithelial lungcarcinoma (A549) cells were obtained from ATCC. A549 cells weremaintained in DMEM with 10% FBS and HuVEC cells were maintained in EGM(includes Basal medium and SingleQuots). For viral infection, A549 cellswere grown in DMEM with 2% FBS, 0.1 nM NEAA and 1%penicillin/streptomycin and HuVEC cells were grown in EGM medium.Viruses were quantitated as previously described.

To determine biological activity of the TK protein, the in vitrocytotoxicity of the virus was assayed with and without addition ofgancyclovir (GCV), a compound that is converted by thymidine kinase intoa triphosphate analog capable of interfering with both cellular andviral replication. Cells were seeded at a density for each cell linethat generated a confluent monolayer within 24 hr. These densely seededcells were allowed to grow for 2 additional days prior to exposure tothe ColoAd1/PL30/TK/TK virus. Infections of both cell lines were carriedout in quadruplicate with serial three-fold dilutions of the virusesstarting at a particle per cell ratio of 100 and ending at a particleper cell ratio of 0.0152. For each cell type, infections were with orwithout GCV (20 uM, added at 24 hours post infection). Infected cellswere incubated at 37° C. for 8 days post infection and cell viabilitywas measured by the MTS assay. Mock-infected cells with or without GCVaddition established the 100% survival point for the given assay.

In HuVec cells (see FIG. 6A), there is no difference in cell killingwith or without GCV addition at the lower viral dose range (0.0152vp/cell to 1.23 vp/cell), but at a higher viral dose range (3.7 vp/cellto 100 vp/cell), addition of GCV prevents cell killing when compared toinfection alone. This is explained by the fact that at higher viraldoses (11, 33 and 100 vp/cell) in the absence of GVC, the level ofinitial infection is high enough to result in significant killing ofcells due to rounds of viral infection and bursting. In the presence ofGCV, the thymidine kinase expressed from the TK promoter within theColoAd1/PL30/TK/TK virus is inhibited and results in death of theinfectious centers, which prevents the viral infection and burstingrounds needed for a significant cell kill. In A549 cells, addition ofGCV significantly protects against cell killing at all doses of viralinfection. The significant killing at all vp/cell (as compared to theHuVEC cells) is due to the fact that the virus replicates much faster inthe A549 cells than in HuVec cells, allowing a much greater number ofrounds of infection and bursting in the absence of GCV.

All patents and publications and patents mentioned in the abovespecification are herein incorporated by reference. While the presentinvention has been described with reference to the specific embodimentsthereof, it should be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the true spirit and scope of the invention. In addition,many modifications may be made to adapt a particular situation,material, composition of matter, process, process step or steps, to theobjective, spirit and scope of the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto.

1. A method of identifying functional insertion sites within the genomeof a replication competent target virus, said method comprising thesteps of: (a) mixing the genomic DNA of said target virus with a donorDNA comprising a transposon under conditions that allow transposition,wherein said transposon comprises at least one genetic element ofinterest and inserts into said viral genome in a non-biased manner; and(b) isolating replication competent viruses from said step (a) whichexpress said genetic element of interest.
 2. The method of claim 1,wherein said isolation step comprises assaying for expression of saidgenetic element of interest in animal cells transfected with saidreplication competent viruses.
 3. The method of claim 1, wherein saidtarget virus is an animal virus.
 4. The method of claim 3, wherein saidanimal virus is oncolytic.
 5. The method of claim 4, wherein said animalvirus is adenovirus, VSV, NDV, HSV, or vaccinia virus.
 6. The method ofclaim 5, wherein said adenovirus is Ad5.
 7. The method of claim 1,wherein said genetic element is a gene which encodes a reportermolecule, a therapeutic protein or an RNA molecule.
 8. The method ofclaim 7, wherein said reporter molecule is GFP, LacZ, renilla luciferaseor firefly luciferase.
 9. The method of claim 8, wherein saidtherapeutic protein is an immunomodulatory protein, an antibody, asymporter or a pro-drug converting enzyme.
 10. The method of claim 1,wherein said transposon further comprises an expression element, whereinsaid element is located upstream from, and is operably linked to, saidgenetic element of interest.
 11. The method of claim 10, wherein saidexpression element is a eukaryotic promoter.
 12. The method of claim 11,wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase,ubiquitin or actin promoter.
 13. The method of claim 12, wherein saidexpression element is a eukaryotic splice acceptor sequence.
 14. Themethod of claim 13, wherein said splice acceptor sequence is SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO:
 3. 15. The method of claim 1, wherein saidtransposon is a transposon which employs an ATP-utilizing regulatoryproteins.
 16. The method of claim 15, wherein said transposon is a Tn-7based transposon.
 17. The method of claim 1, wherein said transposonfurther comprises a selectable gene.
 18. The method of claim 17, whereinsaid selectable gene is an antibiotic resistance gene, a methotrexateresistance gene, or a G418 resistance gene.
 19. The method of claim 1,wherein said donor DNA is a plasmid.
 20. The method of claim 19, whereinsaid donor DNA further comprises an origin of replication.
 21. Themethod of claim 1, wherein said target virus genomic DNA is present in aplasmid.
 22. The method of claim 21, wherein said target virus genomicDNA is flanked by restriction enzyme sites.
 23. The method of claim 22,wherein said restriction enzyme sites are identical.
 24. A method ofidentifying functional insertion sites within the genome of areplication competent target virus, said method comprising the steps of:(a) mixing a plasmid comprising the genomic DNA of said target viruswith a plasmid donor DNA comprising a transposon and an origin ofreplication under conditions that allow transposition, wherein saidtransposon comprises at least one genetic element of interest operablylinked to an expression element and a selectable gene and inserts intosaid target viral genome in a non-biased manner; and (b) isolatingreplication competent viruses from said step (a) which express saidgenetic element of interest.
 25. The method of claim 24, wherein saidisolation step (b) comprises assaying for expression of said geneticelement of interest in animal cells transfected with said replicationcompetent viruses
 26. The method of claim 24, wherein said target virusgenomic DNA is an animal virus.
 27. The method of claim 26, wherein saidanimal virus is oncolytic.
 28. The method of claim 27, wherein saidanimal virus is an adenovirus, VSV, NDV, HSV, or vaccinia virus.
 29. Themethod of claim 28, wherein said adenovirus is Ad5.
 30. The method ofclaim 24, wherein said genetic element is a gene which encodes areporter molecule, a therapeutic protein or an RNA molecule.
 31. Themethod of claim 30, wherein said reporter gene encodes GFP, LacZ,renilla luciferase or firefly luciferase.
 32. The method of claim 31,wherein said therapeutic protein is an immunomodulatory protein, anantibody, a symporter or a pro-drug converting enzyme.
 33. The method ofclaim 24, wherein said expression element is a eukaryotic promoter or aeukaryotic splice acceptor sequence.
 34. The method of claim 33, whereinsaid eukaryotic promoter is a CMV, SV40, thymidine kinase, ubiquitin oractin promoter.
 35. The method of claim 34, wherein said eukaryoticpromoter is the early SV40 promoter.
 36. The method of claim 33, whereinsaid splice acceptor sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ IDNO:
 3. 37. The method of claim 24, wherein said transposon is transposonwhich employs an ATP-utilizing regulatory protein.
 38. The method ofclaim 37, wherein said transposon is a Tn-7-based transposon.
 39. Themethod of claim 24, wherein said selectable gene is an antibioticresistance gene or a drug resistance gene
 40. The method of claim 24,wherein said target virus genomic DNA is flanked by restriction enzymesites.
 41. The method of claim 40, wherein said restriction enzyme sitesare identical.
 42. A method of identifying functional insertion siteswithin the genome of a replication competent Ad5 virus, said methodcomprising the steps of: (a) mixing a plasmid comprising the genomic DNAof said Ad5 virus with a plasmid donor DNA comprising a Tn-7 basedtransposon and an R6K origin of replication under conditions that allowtransposition, wherein said transposon comprises a gene encoding GFPoperably linked to an expression element and a chloramphenicolresistance gene and wherein said transposon inserts into said target Ad5genome in a non-biased manner; and (b) isolating replication competentAd5 viruses which express GFP from said step (a).
 43. The method ofclaim 42, wherein said isolation step (b) comprises the steps of: (a′)transforming bacterial cells with the mixture of said step (a); (b′)isolating replication competent Ad5 viruses from said transformed cells;(c′) transfecting eukaryotic cells with said Ad5 viruses; and (d′)identifying plaques derived from plating of said cells from said step(c′).
 44. The method of claim 42, wherein said expression element is aeukaryotic promoter or a eukaryotic splice acceptor sequence.
 45. Themethod of claim 44, wherein said eukaryotic promoter is a CMV, SV40,thymidine kinase, ubiquitin or actin promoter.
 46. The method of claim45, wherein said eukaryotic promoter is an early SV40 promoter.
 47. Themethod of claim 44, wherein said splice acceptor sequence is SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO:
 3. 48. A replication competent viruscontaining a functional insertion site identified by the method of claim1, claim 24, or claim
 42. 49. A method of generating a replicationcompetent virus comprising at least one genetic element of interestinserted into a functional insertion site within the genome of saidvirus, said method comprising the steps of: (a) mixing the genomic DNAof a target replication competent virus with a donor DNA comprising atransposon, under conditions that allow transposition, wherein saidtransposon comprises a genetic element of interest and inserts into saidviral genome in a non-biased manner; and (b) identifying productreplication competent viruses formed in said step (a) which express saidgenetic element.
 50. The method of claim 49, wherein said target virusis an animal virus.
 51. The method of claim 50, wherein said animalvirus is oncolytic.
 52. The method of claim 51, wherein said animalvirus is adenovirus, VSV, NDV, HSV, or vaccinia virus.
 53. The method ofclaim 52, wherein said adenovirus is Ad5.
 54. The method of claim 49,wherein said genetic element is a gene which encodes a reportermolecule, a therapeutic protein or an RNA molecule.
 55. The method ofclaim 54, wherein said reporter molecule is GFP, LacZ, renillaluciferase or firefly luciferase.
 56. The method of claim 54, whereinsaid therapeutic protein is an immunomodulatory protein, an antibody, asymporter or a pro-drug converting enzyme.
 57. The method of claim 49,wherein said transposon further comprises an expression element, whereinsaid element is located upstream from, and is operably linked to, saidgenetic element of interest.
 58. The method of claim 57, wherein saidexpression element is a eukaryotic promoter.
 59. The method of claim 58,wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase,ubiquitin or actin promoter.
 60. The method of claim 57, wherein saidexpression element is a eukaryotic splice acceptor sequence.
 61. Themethod of claim 60, wherein said splice acceptor sequence is SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO:
 3. 62. The method of claim 49, whereinsaid transposon is a transposon which employs an ATP-utilizingregulatory proteins.
 63. The method of claim 62, wherein said transposonis a Tn-7 based transposon.
 64. The method of claim 49, wherein saidtransposon further comprises a selectable gene.
 65. The method of claim64, wherein said selectable gene is an antibiotic resistance gene, amethotrexate resistance gene, or a G418 resistance gene.
 66. The methodof claim 49, wherein said donor DNA is a plasmid.
 67. The method ofclaim 66, wherein said donor DNA further comprises an origin ofreplication.
 68. The method of claim 49, wherein said target virusgenomic DNA is present in a plasmid.
 69. The method of claim 68, whereinsaid target virus genomic DNA is flanked by restriction enzyme sites.70. The method of claim 69, wherein said restriction enzyme sites areidentical.